International Journal of Molecular Sciences

Review The Roles of FoxO Factors in Regulation of Bone Cells Function

Xiaoli Ma 1,2,3, Peihong Su 1,2,3 , Chong Yin 1,2,3, Xiao Lin 1,2,3, Xue Wang 1,2,3, Yongguang Gao 1,2,3, Suryaji Patil 1,2,3 , Abdul Rouf War 1,2,3 , Abdul Qadir 1,2,3, Ye Tian 1,2,3,* and Airong Qian 1,2,3,* 1 Lab for Bone Metabolism, Key Lab for Space Biosciences and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi’an 710072, China; [email protected] (X.M.); [email protected] (P.S.); [email protected] (C.Y.); [email protected] (X.L.); [email protected] (X.W.); [email protected] (Y.G.); [email protected] (S.P.); [email protected] (A.R.W.); [email protected] (A.Q.) 2 Research Center for Special Medicine and Health Systems Engineering, School of Life Sciences, Northwestern Polytechnical University, Xi’an 710072, China 3 NPU-UAB Joint Laboratory for Bone Metabolism, School of Life Sciences, Northwestern Polytechnical University, Xi’an 710072, China * Correspondence: [email protected] (Y.T.); [email protected] (A.Q.)

 Received: 13 December 2019; Accepted: 17 January 2020; Published: 21 January 2020 

Abstract: Forkhead box class O family member (FoxOs) are evolutionarily conserved transcription factors for their highly conserved DNA-binding domain. In mammalian species, all the four FoxO members, FoxO1, FoxO3, FoxO4, and FoxO6, are expressed in different organs. In bone, the first three members are extensively expressed and more studied. Bone development, remodeling, and homeostasis are all regulated by multiple cell lineages, including osteoprogenitor cells, chondrocytes, osteoblasts, osteocytes, osteoclast progenitors, osteoclasts, and the intercellular signaling among these bone cells. The disordered FoxOs function in these bone cells contribute to osteoarthritis, osteoporosis, or other bone diseases. Here, we review the current literature of FoxOs for their roles in bone cells, focusing on helping researchers to develop new therapeutic approaches and prevent or treat the related bone diseases.

Keywords: FoxOs; bone cells; ; osteogenesis; osteoclastogenesis; chondrogenesis; bone diseases

1. Introduction Forkhead box class O family member proteins (FoxOs) are evolutionarily conserved transcription factors [1]. FoxOs translate environmental signaling into expression [2], regulating many cellular processes, including cell survival, proliferation, differentiation, , oxidative stress resistance, metabolism, inflammation, and aging [3–8]. In bone, internal and external environmental changes cause an imbalance of the bone dynamic, resulting in loss of bone mass and strength, and increased risk for fractures and morbidity [9,10]. Recent studies with murine models of cell-specific loss- or gain-of-function of FoxOs, have revealed that FoxOs regulate bone cell functions and their intercellular signaling. These controls affect bone development, alter bone mass, and conduce several bone diseases, such as osteoarthritis, age-related or estrogen-deficiency osteoporosis, and so on [10]. Hence, we will firstly introduce the structure and function of the FoxO family. Then, we will review the recent developments about FoxOs in osteoprogenitor cells, osteoblasts, hematopoietic stem cells, osteoclast progenitors, osteoclasts, and chondrocytes, as well as the intercellular signaling among

Int. J. Mol. Sci. 2020, 21, 692; doi:10.3390/ijms21030692 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 692 2 of 22 these bone cells. At the same time, we will also describe the FoxO actions in bone cell biology, including cell survival, self-renewal, cell cycle, proliferation, osteogenesis, osteoclastogenesis, chondrogenesis, autophagy, and apoptosis. All of these will focus on their emerging molecular mechanism, pathological implications, and therapeutic potential for related bone diseases.

2. The FoxOInt. Family J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 2 of 24

In mammalianchondrogenesis, species, autophagy, there and are apoptosis. four members All of these of will the focus FoxO on their family: emerging FoxO1 molecular (FKHR), FoxO3 (FKHRL1), FoxO4mechanism, (AFX), pathological and FoxO6 implications, [11]. and FoxO1 therapeutic and FoxO3potential proteinsfor related arebone greater diseases. than 650 amino acids in size, which2. The are FoxO larger Family than FoxO4 and FoxO6 (nearly 500 amino acids). Although, the FoxO6 gene exhibits major structuralIn mammalian diff species,erences there as comparedare four member to thes of other the FoxO three family: family FoxO1 gene (FKHR), members FoxO3 [11]. The four FoxO (FKHRL1), members FoxO4 share (AFX), obvious and FoxO6 sequence [11]. FoxO1 and FoxO3 and proteins possess are greater four clearlythan 650 diaminofferent functional motifs, whichacids include in size, which a forkhead are larger than domain, FoxO4 nuclearand FoxO6 localization,(nearly 500 amino nuclear acids). Although, export, the and FoxO6 transactivation gene exhibits major structural differences as compared to the other three family gene members [11]. domains (FigureThe four1)[ FoxO12 ,protein13]. All members the fourshare obvious FoxO sequ membersence homology are ubiquitously and possess four expressedclearly different [14 ], including FoxO6 that wasfunctional previously motifs, reportedwhich include to exista forkhead basically domain, in thenuclear brain localization, but has beennuclear more export, recently and discovered in other organstransactivation as well [ 4domains,15]. In (Figure particular, 1) [12,13]. FoxO1, All theFoxO4, four FoxO and members FoxO6 are are ubiquitously more detected expressed in the adipose, [14], including FoxO6 that was previously reported to exist basically in the brain but has been more musculoskeletal,recently and discovered nervous in other tissues, organs as respectively, well [4,15]. In particular, while FoxO3 FoxO1, FoxO4, is more and expressedFoxO6 are more in the stomach, spleen, kidney,detected intestine, in the adipose, and cardiacmusculoskeletal, tissues and [13 nervous]. Nevertheless, tissues, respectively, FoxO1, while FoxO3, FoxO3 is and more FoxO4 are all expressed inexpressed bone cells in the [16 stomach,]. spleen, kidney, intestine, and cardiac tissues [13]. Nevertheless, FoxO1, FoxO3, and FoxO4 are all expressed in bone cells [16].

Figure 1. A schematic diagram of the four different functional motifs of the mammalian FoxO family Figure 1. A schematic diagram of the four different functional motifs of the mammalian FoxO family members (FoxOs).members For(FoxOs). humans, For humans, FoxO1, FoxO1, FoxO3, FoxO3, FoxO4, FoxO4, and and FoxO6 FoxO6 proteins proteins are 655 aa, are 673 655aa, 505 aa, aa, 673 aa, 505 aa, and 492 aa inand size, 492 aa respectively. in size, respectively. The The functional functional doma domainsins are arethe forkhead the forkhead domain, nuclear domain, localization, nuclear localization, nuclear export,nuclear and export, a transactivation and a transactivation domain. domain. In FoxO proteins, the forkhead domain located in the N-terminal portion [17] can recognize two In FoxOresponse proteins, elements: the the forkhead insulin-responsive domain elemen locatedt (5’-(C/A) in the (A/C)AAA(C/T)AA) N-terminal portion and Daf-16 [17 (namely] can recognize two response elements:FoxO) family the member insulin-responsive binding element (5´-GTAAA( element (5’-(CT/C)AA)/A) [12–16,18]. (A/C)AAA(C Although/ FoxO1T)AA) recognizes and Daf-16 (namely both the insulin response element and the Daf-16 family member binding element, it has a higher FoxO) familyaffinity member for the bindinglatter [19]. elementThe transactivation (5´-GTAAA(T domain, located/C)AA) in the [12 C-terminal–16,18]. region Although of the FoxOs, FoxO1 recognizes both the insulincombines response with the cis-regulatory element and sites the of FoxO-target Daf-16 family [17]. member In addition, binding the nuclear element, localization it has a higher affinity for thesequence latter and [19 the]. nuclear The transactivation export sequence are domain,within the C-terminal located inDNA the binding C-terminal domain of region FoxOs, of the FoxOs, indispensable for maintaining the FoxO proteins in the nucleus or in the cytosol, respectively [12]. combines withNuclear the FoxO cis-regulatory proteins, predominantly sites of FoxO-target as transcription genes factors, [17 interact]. In addition, with DNA theand nuclearpartner localization sequence andproteins the nuclear to modulate export the transcription sequence of arenumerously within specific the C-terminal target genes [17]. DNA binding domain of FoxOs, indispensable forResponding maintaining to a variety the of FoxO external proteins or internal in stimuli the nucleus (including or starvation, in the cytosol,insulin, growth respectively [12]. factors, hormones, cytokines, and oxidative stress), FoxOs act as transcription factors and rarely as Nuclear FoxOco-regulators, proteins, predominantlyeither activated asor transcriptioninactivated by factors, post-translational interact with modification DNA and (like partner proteins to modulatephosphorylation, the transcription ubiquitination, of numerously acetylation, specific and methylation) target [9,20]. genes Further, [17]. they are also regulated Respondingby lipopolysaccharide to a variety of (LPS), external tumor or necrosis internal factor stimuli α (TNF- (includingα), and the starvation, interaction with insulin, protein growth factors, partners [21]. These modifications alter the nuclear import and export steps of FoxOs, modify the hormones, cytokines, and oxidative stress), FoxOs act as transcription factors and rarely as co-regulators, either activated or inactivated by post-translational modification (like phosphorylation, ubiquitination, acetylation, and methylation) [9,20]. Further, they are also regulated by lipopolysaccharide (LPS), tumor necrosis factor α (TNF-α), and the interaction with protein partners [21]. These modifications alter the nuclear import and export steps of FoxOs, modify the DNA binding affinity of FoxOs, and alter transcriptional activity for FoxOs’ specific target gene. Therefore, FoxOs take part in regulatory Int. J. Mol. Sci. 2020, 21, 692 3 of 22 networks that ensure tight and timely transcriptional control of proteins involved in proliferation (FoxO1, FoxO3, and FoxO4), cellular differentiation (FoxO1, FoxO3, and FoxO4), apoptosis (FoxO1, FoxO3, and FoxO4), oxidative stress resistance (FoxO1 and FoxO3), metabolism (FoxO1 and FoxO3), inflammation (FoxO1, FoxO3, and FoxO4) and aging (FoxO1, FoxO3, and FoxO4) in mammals [3–8]. What is more, a unifying hypothesis has also highlighted the role of FoxOs as signaling integrators for the maintenance of cell and tissue homeostasis over time and in response to environmental challenges, including metabolic stress, oxidative stress, and growth factor deprivation [9].

3. FoxOs and Bone Cells As compared to other organs in the body, bone is constantly degraded and replaced throughout one’s life. The relatively dynamic bone modeling and remodeling are governed specifically by osteoprogenitor cells, osteoblasts, osteocytes, hematopoietic stem cells, osteoclast progenitors, osteoclasts, and chondrocytes, as well as the complex intercellular signaling among these bone cells. Except for osteocytes, all bone cells were found expressing FoxO1, 3, and 4, as in several other mammalian cell types. Functions of FoxOs in these cells are pivotal for bone development, remodeling, and homeostasis manipulation under physiological and pathological conditions.

3.1. Bidirectional Regulation of FoxOs in Osteoprogenitor Cells FoxOs play important roles in the osteogenesis of osteoprogenitor cells containing mesenchymal stem cells (MSCs), the early precursors of osteoblasts. MSCs are multipotent stromal cells. They give rise to chondrocytes, muscle cells, and adipocytes. Moreover, osteogenic differentiation of MSCs into osteoblasts is managed by an array of specific transcription factors (such as TCF, runt-related transcription factor 2, and activating transcription factor 4) and genes (such as the alkaline phosphatase gene, runt-related transcription factor 2 gene, and osteocalcin gene), leading to a succession of osteoblast phenotypic markers [16]. Therefore, it is not difficult to envision the bidirectional regulation role of FoxOs in the entire complex and sensitive osteogenic process (Table1).

3.1.1. FoxOs Promote Osteogenesis With experiments in vitro, ex vivo, and in vivo, Teixeira et al. have demonstrated FoxO1 as an early positive regulator in the osteogenic differentiation of mesenchymal cells [22]. In mouse embryonic mesenchymal cells (C3H10T1/2 cells), osteogenic stimulants enhance the activity and expression of FoxO1. Similarly, overexpressing FoxO1 significantly increases the expression of osteogenic markers such as runt-related transcription factor 2 (Runx2), alkaline phosphatase (Alp), and osteocalcin (Ocn). Conversely, silencing FoxO1 inhibits the expression of these osteogenic markers, decreases calcification, impairs skeletogenesis and craniofacial development, and especially reduces the size of the embryos as well as bone size in the craniofacial area. In addition, knocking down FoxO1 in mouse embryonic tibia ex vivo has been reported to have similar results as those obtained in vivo, which led to shorter and less mineralized bone [22]. Furthermore, in pre-osteoblastic cells (e.g., MC3T3-E1 cells), the upregulated binding activity of FoxO1 to the Runx2 gene promoter increases the mRNA level of Runx2 and alkaline phosphatase (ALP) activity during the formation of mineralizing nodules. In addition, Runx2 directs pluripotent MSCs to the osteoblast lineage and triggers the expression of major bone matrix protein genes (like type I collagen or Alp) in early progenitors. Then, FoxO1 interacts with Runx2 and could promote osteoblast differentiation [16,23]. Therefore, to some extent, FoxO1 promotes osteoblast differentiation by interacting with Runx2 or its gene promoter (Figure2). Besides, FoxO1 could also bind to the ALP gene promoter [24]. BMP-2-induced FoxO1 transcription increases the reporter activity of an ALP gene promoter construct [25]. Thus, FoxO1 may also promote osteoblast differentiation by interacting with the ALP gene promoter (Figure2). Int. J. Mol. Sci. 2020, 21, 692 4 of 22 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 4 of 24

Figure 2. The diverse function of FoxOs in osteoprogenitor cells. Reactive oxygen species (ROS) can Figure 2. The diverse function of FoxOs in osteoprogenitor cells. Reactive oxygen species (ROS) can trigger FoxOstrigger mediated FoxOs mediated transcription transcription and and enhances enhances thethe binding binding of FoxOs of FoxOs to β-catenin, to β-catenin, thus diverting thus diverting the limited β-cateninthe limited poolβ-catenin from pool transcription from transcription factor factor TCF to to FoxO-mediated FoxO-mediated transcription transcription and thereby and thereby decreases osteoblastogenesis.decreases osteoblastogenesis. But FoxO1 But FoxO1 promotes promotes the the transcription transcription of runt-related runt-related transcription transcription factor factor 2 (Runx2) or alkaline phosphatase (Alp), increasing osteoblastogenesis. 2 (Runx2) or alkaline phosphatase (Alp), increasing osteoblastogenesis. Similarly, it has been reported that global deletion of FoxO1, 3, and 4 in mice leads to a reduction Similarly,in itthe has expression been reportedof Runx2, Osterix, thatglobal and alkaline deletion phosphatase of FoxO1, in osteoblastic 3, and progenitors 4 in mice in leadsvitro [26], to a reduction in the expressionand the of mice Runx2, also display Osterix, a significant and alkaline decrease phosphatase in the number inof osteoblasticcell colony-forming progenitors unit (CFU) in vitro [26], fibroblasts and CFU osteoblasts presented in the bone marrow [21]. That means FoxOs could regulate and the miceosteoblast also display differentiation a significant and proliferation. decrease in the number of cell colony-forming unit (CFU) fibroblasts and CFUOn the osteoblasts other hand, FoxO1, presented as a transcriptional in the bone tran marrows-repressor, [21 inhibits]. That the actions means of FoxOsperoxisome could regulate osteoblast diffproliferator-activatederentiation and proliferation. γ (PPARγ) [27], which activates adipogenesis and suppresses osteoblastogenesis. The deletion of FoxOs increases PPARγ expression [21]. Consistent with these On thefindings, other hand,FoxO1 restrains FoxO1, adipogenesis as a transcriptional by repressing the activity trans-repressor, of the PPARγ promoter inhibits and the by actions of peroxisome proliferator-activatedantagonizing PPARγ’s ability receptor by directlyγ (PPAR binding γto)[ PPAR-response27], which activateselements (PPREs), adipogenesis the promoters and suppresses of PPARγ target genes in pre-adipocytes [28,29]. As aγ result, FoxOs improve bone formation, by osteoblastogenesis.increasing The osteoblast deletion differentiation of FoxOs increases at the expens PPARe of adipocyteexpression differentiation [21]. Consistent from their common with these findings, FoxO1 restrainsmesenchymal adipogenesis progenitors. by repressing the activity of the PPARγ promoter and by antagonizing PPARγ’s ability by directly binding to PPAR-response elements (PPREs), the promoters of PPARγ target genes in pre-adipocytes [28,29]. As a result, FoxOs improve bone formation, by increasing osteoblast differentiation at the expense of adipocyte differentiation from their common mesenchymal progenitors.

Int. J. Mol. Sci. 2020, 21, 692 5 of 22

Table 1. The bidirectional regulation role of FoxO transcription factors (FoxOs) in osteoprogenitor cells.

Functions in Osteoprogenitor FoxOs Effects on Bone Mechanisms Cell/Mice Models References Cell C3H10T1/2 cells with FoxO1 overexpression or silencing; mice osteogenesis differentiation (+); skeletogenesis (+); FoxO1 interacts with with downregulated FoxO1 [22] FoxO1 calcification culture (+) craniofacial development Runx2 promoter (+); expression in developing embryos (+); craniofacial area (+) Runx2, ALP and OCN in vivo/embryonic tibiae ex vivo expression (+) osteoblast differentiation (+); depletion/overexpression of FoxO1 [25] osteoblast proliferation (-) in MC3T3-E1 cells CFU-osteoblasts (+); [21] conditional deletion of FoxO1, 3, Runx2, Osterix, and ALP and 4 in 3-month-old mice oxidative stress (-); osteogenic differentiation (+) [26] expression (+) skeletal homeostasis (+) global deletion of FoxO1, 3, and 4 Adipogenesis (-) PPARγ (-) [21] FoxO1, 3, and 4 in mice mice lacking FoxO1, 3, and 4 in bone mass (-); osteoblast osteoprogenitor cells Wnt/β-catenin signaling bipotential progenitors of [18] numbers (-) proliferation (-) (-); cyclin D1 expression (-) osteoblast and adipocytes (expressing Osterix1) Note: (+) refers to a positive effect and (-) refers to a negative effect. Int. J. Mol. Sci. 2020, 21, 692 6 of 22

3.1.2. FoxOs Repress Osteogenesis In contrast to the above findings, some researchers also indicate FoxOs repress the osteogenic actions by suppressing Wnt signaling in osteoblast progenitors. Conditional knocked-out mice of FoxO1, 3, and 4 in bipotential progenitors of osteoblast and adipocytes showed that osteoblast number and bone mass were increased, but unexpectedly, there were no changes in the redox balance. The increased bone mass rooted in the increased proliferation of osteoprogenitor cells and bone formation, which accounted for the upregulation of Wnt/β-catenin signaling and cyclin D1 expression [18]. In addition, overexpression of FoxO1 reduced the MC3T3-E1 cell number and the number of proliferating cell nuclear antigen (PCNA)-positive cells with little effect on apoptosis, indicating that FoxO1 suppressed preosteoblast proliferation [25]. Moreover, in osteoblast progenitors, FoxOs acts as defense molecules in oxidative stress to regulate osteogenic actions. Excessive accumulation of reactive oxygen species (ROS) generally causes oxidative stress and destroys proteins, lipids, and DNA, even consequentially leading to cell death [30]. As a result of the conditional deletion of FoxO1, FoxO3, and FoxO4 in 3-month-old mice, oxidative stress and osteoblast apoptosis increased in bone, and the number of osteoblasts, the rate of bone formation, and bone mass were all decreased at cancellous or cortical sites [26]. So, we can infer that FoxOs can protect osteogenesis against oxidative stress. However, in osteoblast precursors, FoxOs are induced by the oxidative stress (exemplified by H2O2) to bind with β-catenin to co-activate the target genes’ transcriptions of FoxOs at the expense of Wnt/TCF-mediated transcription and osteoblast differentiation (Figure2)[ 31]. Moreover, in aging mice, the expression of β-catenin/TCF-target genes decreases whereas FoxO-target genes increases in bone, along with an increase in markers of oxidative stress and a decrease in bone formation [31,32]. In line with this idea, fatty acid-binding protein 4 (FABP4)-Wnt10b mice, which express the Wnt10b transgene in marrow, and the mice with a mutation (G171V) in the low-density lipoprotein receptor related protein 5 (LRP5), both reveal enhanced bone mass and no evidence of age-related loss of bone mass or strength [21,33,34]. Similarly, the lipid oxidation may also suppress Wnt signaling by the same mechanism, and result in the decline in osteoblast number and bone formation that occurs with aging. It has been shown that lipoxygenases oxidize polyunsaturated fatty acids to form products, which bind to and activate PPARγ and generate pro-oxidants like 4-hydroxynonenal (4-HNE) [35]. The Almeida group demonstrated that lipid oxidation increases with age in bone [21]. Similar to H2O2, the 4-HNE also activates FoxOs that in turn attenuate β-catenin/TCF-mediated transcription [36]. Hence, it also enhances PPARγ levels for the suppression of TCF-mediated transcription to PPARγ expression [37,38]. On the other hand, the PPARγ bind to β-catenin and induce β-catenin degradation [36,39], thereby further diminishing β-catenin/TCF-mediated transcription. Therefore, at least in part, it is reasonable to understand that the bone loss with aging might arise from the repressive effect of ROS on Wnt/β-catenin signaling via FoxO activation. Responding to oxidative stress, FoxOs also restrain other main osteogenic signaling pathways (Hedgehog signaling and Mitogen-activated protein kinases signaling) while boosting adipogenic signaling pathways [40]. Meanwhile, they also promote expression of , which also increases adipogenic differentiation [40]. It is known that adipogenic differentiation is antagonistic to the osteogenic differentiation. Thus, FoxOs may also accelerate bone loss with aging by enhancing adipogenic differentiation. Obviously, in response to oxidative stress, FoxOs play an essential role to maintain skeletal homeostasis in osteoprogenitor cells. Taken together, in osteoblast progenitor cells, FoxOs have complicated and contradictory features (Figure2). There is no doubt that FoxOs are important in defense during oxidative stress and prevent cell death triggered by ROS, thus promoting osteogenesis. On one hand, MSCs have relatively high expression of antioxidant-like glycolysis and low ROS levels to keep quiescent [40], but a high ROS level may act as an intracellular signal to drive MSCs to exit from quiescence and result in the activation of a genetic program triggering osteoblast precursors commitment [16]. So, at the initial stage of differentiation, the switching from FoxO-mediated transcription to Wnt/TCF-mediated transcription Int. J. Mol. Sci. 2020, 21, 692 7 of 22 may benefit osteogenesis. On the other hand, with the progress of osteogenic differentiation, FoxO1 may directlyInt. participateJ. Mol. Sci. 2020 in, 21 osteoblastogenesis, x FOR PEER REVIEW promotion by binding to the ALP and Runx2 gene promoters2 of 24 or directly interacting with Runx2, as well as maintaining a low level of ROS to protect cells from oxidativedifferentiation, stress. Besides, FoxO1 FoxOsmay directly decrease participate ROS levels in osteoblastogenesis to restrain adipogenic promotion differentiation. by binding to Therefore, the the specificALP and role Runx2 of FoxOs gene promoters may depend or directly on the interactin differentiationg with Runx2, stage as and well ROS as maintaining level of osteoprogenitor a low level of ROS to protect cells from oxidative stress. Besides, FoxOs decrease ROS levels to restrain cells. Further studies are still needed to fully reveal the multiple functions of FoxOs in osteoprogenitor adipogenic differentiation. Therefore, the specific role of FoxOs may depend on the differentiation cells at a subtle stage during osteogenic differentiation. stage and ROS level of osteoprogenitor cells. Further studies are still needed to fully reveal the multiple functions of FoxOs in osteoprogenitor cells at a subtle stage during osteogenic 3.2. FoxOs Improve Osteoblasts Function differentiation. The effects of FoxOs on osteoblasts are also complicated and likely to depend upon specific conditions (Table3.2.2). Nevertheless,FoxOs Improve mostOsteoblasts of the Function literature support the positive effect of FoxOs in osteoblasts. On one hand, FoxOs regulateThe effects osteoblast of FoxOs numbers on osteoblasts significantly are by also improving complicated antioxidant and likely defense to depend (Figure upon3). Conditionally specific overexpressedconditions FoxO3(Table 2). in Nevertheless, osteoblasts decreasesmost of the ROS literature and further support inhibits the positive osteoblast effect of apoptosis FoxOs in with reducingosteoblasts. phosphorylation On one hand, of FoxOs p66Shc. regulate Consistent osteoblast with numbers this, significantly conditional by deletion improving of antioxidant all three FoxO membersdefense (FoxO1 (Figure/3/ 4)3). in Conditionally 3-month-old overexpressed mice decrease FoxO3 osteoblast in osteoblasts numbers decreases due to osteoblastROS and further apoptosis inhibits osteoblast apoptosis with reducing phosphorylation of p66Shc. Consistent with this, with arising oxidative stress. FoxOs inactivation also inhibits osteoblastogenesis [26]. Moreover, conditional deletion of all three FoxO members (FoxO1/3/4) in 3-month-old mice decrease osteoblast osteoblast-specific deletion of FoxO1 leads to increased oxidative stress, which decreases osteoblast numbers due to osteoblast apoptosis with arising oxidative stress. FoxOs inactivation also inhibits numbersosteoblastogenesis and bone mass [26]. [41 Moreover,]. More osteoblast-specific precisely, the FoxO1 deletion inactivation of FoxO1 leads weakens to increased its interaction oxidative with activatingstress, transcription which decreases factor osteoblast 4 (ATF4), reducingnumbers theand target bone gene mass binding [41]. More activity precisely, of FoxO1 the and FoxO1 impeding aminoinactivation acid import weakens and its protein interaction synthesis with activating through transcription ATF4 (Figure factor3). 4 Therefore, (ATF4), reducing as transcriptional the target regulationgene binding genes of activity FoxO1, of antioxidantFoxO1 and impeding gene (such amino as Sod2 acidand import ) and protein expression synthesis decreases, through increasingATF4 the ROS(Figure level 3). and Therefore, exerting as harmful transcriptional effects on regulation osteoblast genes survival. of FoxO1, And antioxidant with FoxO1 gene deletion, (such as the Sod2 blocked proteinand synthesis catalase) breaks expression the redox decreases, balance increasing by improving the ROS the expressionlevel and exerting of p19ARF harmful (an alternate effects readingon frameosteoblast protein productsurvival. And of the with CDKN2A FoxO1 deletion, locus inthe mice) blocked and protein p16 andsynthesis downstream breaks the activation redox balance of their by improving the expression of p19ARF (an alternate reading frame protein product of the CDKN2A target protein , repressing the cell cycle of the osteoblasts and finally decreasing osteoblast numbers. locus in mice) and p16 and downstream activation of their target protein p53, repressing the cell cycle Additionally,of the osteoblasts the blocked and protein finally synthesisdecreasing also osteob suppresseslast numbers. glutathione Additiona accumulation,lly, the blocked which protein induces oxidativesynthesis stress also [41 ].suppresses Taken together, glutathione FoxOs accumulation, enhance the which activity induces of osteoblasts oxidative andstress protect [41]. Taken these cells throughtogether, the induction FoxOs enhance of antioxidants the activity (Figure of osteoblasts3). and protect these cells through the induction of antioxidants (Figure 3).

FigureFigure 3. FoxOs’ 3. FoxOs’ function function in osteoblasts. in osteoblasts. FoxOs’ FoxOs’ transcriptional transcriptional activity activity reduces reduces ROS ROS levels levels and improvesand osteoblastimproves survival. osteoblast The interaction survival. The between interaction FoxO1 between and activating FoxO1 and transcription activating transcription factor 4 (ATF4) factor maintains 4 osteoblast(ATF4) normal maintains proliferation osteoblast bynormal preventing proliferation ROS by or preventing enhancing ROS protein or enhancing synthesis. protein In addition, synthesis. FoxO1 suppressesIn addition, the interaction FoxO1 suppresses between thethe Runx2interaction and betweenBglap2 (osteocalcin the Runx2 and gene) Bglap2 promoters, (osteocalcin directly gene) bind to

the Bglap2promoters, promote directly r, or bind both to to the inhibit Bglap2 osteocalcin promote r, or (OCN) both to expression. inhibit osteocalcin (OCN) expression.

However, FoxO1 can restrain the energy metabolism function of osteoblasts through inhibiting osteocalcin (OCN) expression. Furthermore, FoxO1 can suppress the interaction between the Runx2 and Bglap2 (OCN gene) promoters, directly bind to the Bglap2 promoter, or both to reduce the transcriptional activity of Bglap2 [16,42–44]. Generally, OCN also improves bone mineralization, calcium ion homeostasis, and prevents excessive mineralization. Thus, it is possible that FoxO1 mediates the dual roles of OCN in osteogenesis. But, obviously the effect of FoxO1 on Runx2 activity

Int. J. Mol. Sci. 2020, 21, 692 8 of 22

Table 2. Bone metabolism regulatory functions of FoxO transcription factors in osteoblast.

FoxOs Effects on Bone Functions in Osteoblast Mechanisms Cell/Mice Models References osteoblast number (+) through osteoblastogenesis (+); FoxO1, 3, bone mass (+); bone osteoblast number (+); osteoblast conditional deletion of FoxO1, osteoblast apoptosis (-) through a cell-autonomous and 4 formation rate (BFR) (+) apoptosis (-); oxidative stress (-) 3, and 4 in 3-month-old mice mechanism that enhances oxidative stress osteoblast numbers (+); oxidative ROS activates the p53 signaling cascade, inducing cell [26] FoxO1 BFR (+); bone volume (+) FoxOs deletion mice in bone stress (-) cycle arrest and limiting osteoblast proliferation. mice overexpressing FoxO3 vertebral bone mass (+); osteoblast number (+), osteoblast FoxO3 ROS (-); phosphorylation of p66 Shc (-) under the control of the BFR (+) apoptosis (-), oxidative stress (-) osteocalcin promoter FoxO1 interacts with ATF4 and promotes amino acid bone mass (+), BFR (+) osteoblast proliferation (+), import to favor the protein synthesis, such as FoxO1 deletion in mice from FoxO1 [41] bone volume (+) oxidative stress (-) glutathione. FoxO1 reduces ROS, activating a p53 collagen1a1 expressing cells signaling cascade, then promoting cell cycle. Int. J. Mol. Sci. 2020, 21, 692 9 of 22

However, FoxO1 can restrain the energy metabolism function of osteoblasts through inhibiting osteocalcin (OCN) expression. Furthermore, FoxO1 can suppress the interaction between the Runx2 and Bglap2 (OCN gene) promoters, directly bind to the Bglap2 promoter, or both to reduce the transcriptional activity of Bglap2 [16,42–44]. Generally, OCN also improves bone mineralization, calcium ion homeostasis, and prevents excessive mineralization. Thus, it is possible that FoxO1 mediates the dual roles of OCN in osteogenesis. But, obviously the effect of FoxO1 on Runx2 activity in osteoblasts is opposite in mesenchymal progenitors, and the mechanism is not well known. In addition, accompanied by immune response, FoxO1 also potentially contributes to immune-mediated inhibition of bone formation through accelerating osteoblast apoptosis [45]. Therefore, among the three FoxO proteins, FoxO1 is mainly required for redox balance and proliferation of osteoblasts and thereby to regulate bone formation.

3.3. FoxOs Positively Regulate Hematopoietic Activities Hematopoietic stem cell (HSC) is an osteoclast progenitor. FoxOs maintain quiescence and Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 1 of 24 self-renewal of HSCs mainly through transcriptional regulation of cell cycle arrest and oxidative stress resistance3.4. (Figure FoxOs4 Regulatea). The Osteoclastogenesis loss of FoxO3 in HSCs not only leads to oxidative DNA damage in HSCs by interfering in the base excision repair pathway [46], but also damages the HSC pool with regulating the cell cycle through3.4.1. FoxOs ROS-independent Activate Osteoclastogenesis modulations of the tumor suppressor protein ataxia telangiectasia mutated (ATM)Complex and p16INK4a osteoclastogenesis and ROS-mediated is governed by activation macrophage of p19ARFcolony-stimulating/p53/p21CIP1 factor/ WAF11 (M-CSF)/Sdi1 tumor suppressorand pathways receptor activator [47]. Likewise, of the NF-κB conditional (RANK) ligand deletion (RANKL), of FoxO1,as well as FoxO3,other cytokines and FoxO4 secreted in by the adult mice hematopoieticosteoblasts and system osteocytes decreases that thecontrol expression various st ofeps antioxidant of the osteoclast enzyme differentiation genes, including process, catalase, including precursor proliferation, commitment, differentiation, and maturation [49]. In osteoclast glutathioneprecursors, peroxidase M-CSF 1 can (Gpx-1 stimulate), and expression superoxide of RANK dismutase, which promotesSod1, Sod2 osteoclast, and differentiationSod3, resulting [50]. in ROS over-accumulation.FoxO1 deletion The in followingbone marrow physiologic macrophages oxidative (BMMs) decreases stress increasesM-CSF-induced HSCs’ RANK apoptosis expression and drives HSCs outand of quiescence migration ofinto osteoclast the cell precursors cycle. Meanwhile,[51]. RANKL indirectly HSCs repopulating induces nuclear activity factor haveof activated been long-termT impaired.cells All 1 of (NFATc1) these imply of osteoclast that FoxOs precursors, deficiency dramati enforcescally facilitating HSCs intoosteoclast terminal differentiation differentiation [52]. at the FoxO1 deletion in osteoclast precursors reduces expression and nuclear localization of NFATc1 [51]. expense of self-renewal. Finally, combined FoxOs’ deletion markedly decreases HSCs’ population and In line with this, conditional deletion of FoxO1, FoxO3, and FoxO4 in 3-month-old mice reduces the also expansesexpression myeloid of osteoclastogenesis lineage [48]. Therefore, related factors FoxOs lik aree the likely calcitonin to serve receptor, as protectors tartrate-resistant of HSCs, acid decreasing the numberphosphatase of osteoclast (TRAP), progenitor and cathepsin cells K to [26]. some Therefore, degree FoxOs (Table should3). activate osteoclastogenesis by mediating the effect of M-CSF or RANKL on osteoclast precursors (Figure 4b).

(a) (b)

Figure 4. FigureFoxOs 4. FoxOs regulate regulate osteoclastogenesis osteoclastogenesis and bone bone resorption. resorption. (a) FoxOs (a) promote FoxOs survival promote and survival self- and self-renewalrenewal of hematopoietic of hematopoietic stem stem cells cells byby enhancing the the expression expression of antioxidant of antioxidant enzymes. enzymes. Moreover, Moreover, FoxO3 also regulates expression of the ATM tumor suppressor to decrease ROS level and p16INK4a FoxO3 also regulates expression of the ATM tumor suppressor to decrease ROS level and p16INK4a expression, thereby maintaining cell cycling and self-renewal. (b) FoxO1 stimulates expression,osteoclastogenesis thereby maintaining may by cellmediating cycling theand effect self-renewal. of M-CSF or RANKL (b) FoxO1 on osteoclast stimulates precursors. osteoclastogenesis But may by mediatingconversely, the FoxOs effect restrain of M-CSF osteoclastogenesis, or RANKL onwhich osteoclast mediated precursors. the regulation But of conversely,Akt or Sirt1. ATM FoxOs = restrain osteoclastogenesis,ataxia telangiectasia which mediated mutated, p16INK4a the regulation = p16 (a oflso Akt known or Sirt1. as multiple ATM tumor= ataxia suppressor telangiectasia 1), Akt =mutated, p16INK4aProtein= p16 kinase (also knownB, Sirt1 as= Sirtuin multiple 1, RANK tumor = suppressorreceptor activator 1), Akt of =theProtein NF-κB, kinaseRANKL B, = Sirt1receptor= Sirtuin 1, activator of the NF-κB ligand, M-CSF = macrophage colony-stimulating factor 1, NFATc1 = nuclear RANK = receptor activator of the NF-κB, RANKL = receptor activator of the NF-κB ligand, M-CSF = factor of activated T cells 1. macrophage colony-stimulating factor 1, NFATc1 = nuclear factor of activated T cells 1. 3.4.2. FoxOs Suppress Osteoclastogenesis Other researchers have demonstrated that FoxOs negatively mediate RANKL-induced osteoclast formation. Overexpressing FoxO3 in monocyte/macrophage lineage cells decreases several resorption markers and increases bone mass [21]. The phosphoinositide 3-kinase (PI3K)/Akt pathway is the main negative regulator of FoxOs activity [6]. In osteoclast precursors, RANKL activates Akt signaling (like Akt/PI3K signaling) to promote proliferation, differentiation, and also attenuate apoptosis [50,53,54]. Along with these evidence, the deletion of FoxO1, FoxO3, and FoxO4 or overexpression of FoxO3 or mitochondria-targeted catalase in osteoclast precursors further elucidate that FoxOs upregulate the H2O2-inactivating enzyme catalase, and attenuates H2O2 accumulation, partly restraining the RANKL–Akt mediated osteoclastogenesis action [55]. Besides, Tan et al. have indicated that FoxO1 antagonizes ROS generation and thereby represses Akt, mitogen-activated protein kinases (MAPKs), and nuclear factor kappa-B (NF-κB), including the activated pathways of

Int. J. Mol. Sci. 2020, 21, 692 10 of 22

Table 3. Bone metabolism regulatory functions of FoxO transcription factors in hematopoietic stem cell.

Effects on Functions in Hematopoietic FoxOs Mechanisms Cell/Mice Models References Bone Stem Cell oxidative DNA damage in FoxO3 ROS (-); the base excision repair pathway (+) [47] HSC and progenitor cell (-) / ROS-independent modulations of ATM and mice model with FoxO3− − in HSC HSC quiescence (+); HSC p16INK4a and ROS-mediated activation of FoxO3 [46] G2/M transition (-) p53/p21CIP1/WAF1/Sdi1 tumor suppressor pathways (-) bone mass mice overexpressing FoxO3 in FoxO3 osteoclast number (-) [21] (+) monocyte/macrophage lineage cells partly by impairing detoxification of ROS, FoxO1, 3, HSC quiescence (+); HSC FoxOs decreases HSC apoptosis and conditional deletion of FoxO1, 3, and 4 in the [48] and 4 compartment survival (+) HSC-specific entry into the S/G2/M and G1 adult mice hematopoietic system phases of the cell cycle Int. J. Mol. Sci. 2020, 21, 692 11 of 22

3.4. FoxOs Regulate Osteoclastogenesis

3.4.1. FoxOs Activate Osteoclastogenesis Complex osteoclastogenesis is governed by macrophage colony-stimulating factor 1 (M-CSF) and receptor activator of the NF-κB (RANK) ligand (RANKL), as well as other cytokines secreted by osteoblasts and osteocytes that control various steps of the osteoclast differentiation process, including precursor proliferation, commitment, differentiation, and maturation [49]. In osteoclast precursors, M-CSF can stimulate expression of RANK, which promotes osteoclast differentiation [50]. FoxO1 deletion in bone marrow macrophages (BMMs) decreases M-CSF-induced RANK expression and migration of osteoclast precursors [51]. RANKL indirectly induces nuclear factor of activated T cells 1 (NFATc1) of osteoclast precursors, dramatically facilitating osteoclast differentiation [52]. FoxO1 deletion in osteoclast precursors reduces expression and nuclear localization of NFATc1 [51]. In line with this, conditional deletion of FoxO1, FoxO3, and FoxO4 in 3-month-old mice reduces the expression of osteoclastogenesis related factors like the calcitonin receptor, tartrate-resistant acid phosphatase (TRAP), and cathepsin K[26]. Therefore, FoxOs should activate osteoclastogenesis by mediating the effect of M-CSF or RANKL on osteoclast precursors (Figure4b).

3.4.2. FoxOs Suppress Osteoclastogenesis Other researchers have demonstrated that FoxOs negatively mediate RANKL-induced osteoclast formation. Overexpressing FoxO3 in monocyte/macrophage lineage cells decreases several resorption markers and increases bone mass [21]. The phosphoinositide 3-kinase (PI3K)/Akt pathway is the main negative regulator of FoxOs activity [6]. In osteoclast precursors, RANKL activates Akt signaling (like Akt/PI3K signaling) to promote proliferation, differentiation, and also attenuate apoptosis [50,53,54]. Along with these evidence, the deletion of FoxO1, FoxO3, and FoxO4 or overexpression of FoxO3 or mitochondria-targeted catalase in osteoclast precursors further elucidate that FoxOs upregulate the H2O2-inactivating enzyme catalase, and attenuates H2O2 accumulation, partly restraining the RANKL–Akt mediated osteoclastogenesis action [55]. Besides, Tan et al. have indicated that FoxO1 antagonizes ROS generation and thereby represses Akt, mitogen-activated protein kinases (MAPKs), and nuclear factor kappa-B (NF-κB), including the activated pathways of RANKL-induced osteoclastogenesis [56]. What is more, the inhibitory effect of FoxO1 on osteoclastogenesis is partially mediated by suppression of transcription factor [57], which has been shown to reduce ROS production by inhibiting mitochondrial function [4,58,59]. Taken together, phosphorylation and inhibition of FoxO activity might partially mediate the positive effects of RANKL-induced Akt in osteoclast precursors (Figure4b). Beyond that, RANKL also prevents FoxO activity via acetylation. But Sirtuin 1 (Sirt1) deacetylates FoxO1, FoxO3, and FoxO4, and thereby stimulates FoxO-mediated transcription of catalase and hemeoxygenase-1 (HO-1). In osteoclast precursors, HO-1 attenuates mitochondrial oxidative phosphorylation and ATP production [60] and, along with catalase, reduce H2O2 levels. Both low ATP and H2O2 levels repress osteoclastogenesis [61]. Therefore, Sirt1 contributes to the anti-osteoclastogenic effects of FoxOs (Figure4b). Similarly, mammalian sterile 20-like (Mst) kinase 1 (Mst1) is also a FoxO activating kinase and might facilitate osteoclast apoptosis via activation of FoxO-mediated transcription. When osteoclasts apoptosis is induced by serum withdrawal, staurosporine or bisphosphonates, Mst1 is identified as a key intermediate [62]. In detail, these apoptosis-inducing treatments promote the caspase cleavage of Mst1 into a positive 34 kDa species that keeps the catalytic domain and conduces to phosphorylate FoxOs [63]. However, it remains unknown whether FoxOs mediate the actions of Mst1 on osteoclast generation and survival.

3.4.3. FoxOs, Expressed in Osteoblasts, Indirectly Repress Osteoclastogenesis Unexpectedly, FoxOs also repress osteoclastogenesis indirectly via osteoblasts. In mice, conditional deletion of FoxO1 or overexpression of FoxO3 in osteoblasts results in increased or reduced osteoclast Int. J. Mol. Sci. 2020, 21, 692 12 of 22 numbers, respectively [26]. Hence, activated FoxOs in osteoblastic cells also reduce osteoclast numbers. Osteoprotegerin (OPG), largely produced by osteoblasts, can bind and neutralize RANKL and block osteoclast formation in vitro and bone resorption in vivo [64]. Targeted deletion of FoxO1 in osteoblasts in mice diminishes OPG expression in bone. And FoxO1 activated or not in osteoblasts, in vitro, increases or decreases OPG, respectively [42]. Therefore, FoxO1 may control osteoclast numbers by increasing the OPG expression of osteoblasts. However, mice with overexpressed FoxO3 in osteoblasts does not exhibit any altering OPG levels in bone, hence indicating that FoxO3 might regulate osteoclast numbers independent of OPG [26]. Above all, in osteoclastogenesis, a relatively high ROS level increases HSC apoptosis and drives HSCs out of quiescence into the cell cycle and further terminal differentiation at the expense of self-renewal. But FoxOs fight against oxidative stress and thus protect HSCs from osteoclast differentiation. Moreover, in osteoclast precursor cells, relatively high ROS level promotes osteoclastogenesis [65]. But FoxOs also attenuate osteoclastogenesis or osteoclast activity largely for their oxidative resistance role, which is activated by Sirt1 or Mst1 and suppressed by Akt. In contrast, some researchers have suggested that FoxO1 stimulates osteoclastogenesis by mediating the effects of MSC-F or RANKL on osteoclast precursors. Nevertheless, ROS level is important for FoxOs for controlling osteoclastogenesis; the majority of the literature demonstrates that FoxOs negatively regulate osteoclastogenesis (Figure4; Tables3 and4). Int. J. Mol. Sci. 2020, 21, 692 13 of 22

Table 4. Bone metabolism regulatory functions of FoxO transcription factors in osteoclast and progenitor cells.

Functions in Osteoclast FoxOs Effects on Bone Mechanisms Cell/Mice Models References and Progenitor Cell FoxO1,3,4f/f; LysM-Cre C57BL/6 osteoclast progenitor FoxOs upregulate the H O -inactivating FoxO1, 3, 2 2 mice; transgenic C57BL/6 mice: bone resorption (-) proliferation (-); osteoclast enzyme catalase and attenuates H O [55] and 4 2 2 mitochondria-targeted catalase in lifespan (-) accumulation osteoclasts osteoclast progenitor FoxOs increase the expression of the FoxO1, 3, conditional deletion of FoxO1, 3, bone mass (+); BFR (+) numbers (-); osteoclast osteoclast-specific markers like the calcitonin and 4 and 4 in 3-month-old mice [26] apoptosis (-) receptor, TRAP, and cathepsin K. osteoclast progenitor mice overexpressing FoxO3 under vertebral bone mass (+); FoxO3 numbers (-); osteoclast the control of the BFR (+) numbers (-) osteocalcin promoter bone resorption (-); FoxO1 deletion in mice from FoxO1 [41] osteoclast surface (-) collagen1a1 expressing cells MAPKs, NF-κB and AP-1 (-); osteoclast differentiation bone marrow mononuclear cells; FoxO1 MYC activity (-); [57] (-); osteoclast activity (-) RAW264.7 cells ROS (-); FoxO1 activates osteoclast formation by osteoclastogenesis (+); mediating the effect of RANKL on NFATc1 LyzM.Cre+FoxO1 L/L mice; BMMs osteoclastogenesis of osteoclast activity (+); and several downstream effectors; FoxO1 FoxO1 or RAW264.7 cells transfected with [51] calvarial bone (+) osteoclast precursor deletion or knockdown reduces M-CSF FoxO1 siRNA migration (+) induced RANK expression and migration of osteoclast precursors. Note: TRAP = Tartrate-resistant acid phosphatase, AP-1 = activator protein 1, NFATc1 = nuclear factor of activated T cells 1. Int. J. Mol. Sci. 2020, 21, 692 14 of 22

3.5. FoxOs Facilitate Chondrogenesis In human and mice models, FoxO1 and FoxO3 expressions are dramatically decreased in the superficial zone of cartilage regions during joint aging. There are obvious FoxO phosphorylation and cytoplasmic localization in chondrocyte clusters of osteoarthritis (OA) cartilage [66]. Furthermore, combined deletion of FoxO1, 3, and 4 in mice growth plate chondrocytes (Col2Cre-FoxO1, 3, and 4 3.5. FoxOs Facilitate Chondrogenesis triple knockout; Col2Cre-TKO) and Col2Cre-FoxO1 single knockout results in the overall body and tail length increasing,In human and the mice articular models, cartilage FoxO1 and becoming FoxO3 expressions thicker are in dramatically young mice, decreased and severe in the skeletal superficial zone of cartilage regions during joint aging. There are obvious FoxO phosphorylation and deformitiescytoplasmic occurring localization at older stages,in chondrocyte such as clusters hyperkyphosis of osteoarthritis or OA-like (OA) cartilage changes [66]. in cartilage,Furthermore, synovium, and subchondralcombined bone. deletion These of FoxO1, abnormalities 3, and 4 in mice are associatedgrowth plate with chondrocytes the distinctly (Col2Cre-FoxO1, elongated 3, and hypertrophic 4 zone of thetriple growth knockout; plate Col2Cre-TKO) or the increased and Col2Cre-FoxO1 height of the si proliferativengle knockout results zone ofin the overall proximal body tibial and growth plate [67,68tail]. length Noticeably, increasing, FoxOs the arearticular indispensable cartilage becoming for the thicker growth, in development,young mice, and and severe health skeletal of cartilage deformities occurring at older stages, such as hyperkyphosis or OA-like changes in cartilage, (Table5). synovium, and subchondral bone. These abnormalities are associated with the distinctly elongated Chondrogenesishypertrophic zone begins of the with growth the plate mesenchymal or the increase cellsd height differentiating of the proliferative to chondrocytes, zone of the proximal which then proliferate,tibial hypertrophically growth plate [67,68]. differentiate, Noticeably, and FoxOs terminally are indispensable be apoptotic. for the FoxO3A growth,expression development, is upregulatedand during chondrogenesishealth of cartilage of human(Table 5). primary MSCs, blocking the hypertrophic differentiation and promoting apoptosis [69].Chondrogenesis In human chondrocytes, begins with the mesenchymal downregulation cells differentiating of FoxO transcription to chondrocytes, factors which with then siFoxO1 proliferate, hypertrophically differentiate, and terminally be apoptotic. FoxO3A expression is and siFoxO3upregulated reduces during cell viability chondrogenesis in response of human to ROS. primary The reduced MSCs, antioxidantblocking the proteins,hypertrophic such as the ROS scavengers,differentiation GPX-1, and andpromoting catalase, apoptosis lead to[69]. an In increase human chondrocytes, in intracellular downregulation oxidative stress.of FoxOAnd the simultaneouslytranscription raised factors autophagy-related with siFoxO1 and proteins,siFoxO3 reduces such cell as microtubule-associatedviability in response to ROS. proteinThe reduced 1A/ 1B-light chain 3 (LC3)antioxidant and Beclin1, proteins, give such rise as tothe ROS-induced ROS scavengers, apoptosis GPX-1, [and70]. catalase, Furthermore, lead to similaran increase to the in strongly intracellular oxidative stress. And the simultaneously raised autophagy-related proteins, such as reduced sirtuin 1 (SIRT-1) expression in OA cartilage [71], FoxO1 or FoxO1+3 (FoxO1 and FoxO3) microtubule-associated protein 1A/1B-light chain 3 (LC3) and Beclin1, give rise to ROS-induced downregulationapoptosis also [70]. suppresses Furthermore, SIRT-1; similar thisto the regulates strongly oxidativereduced sirtuin stress 1 (SIRT-1) and autophagy expression by in modulatingOA the signalingcartilage of FoxO [71], FoxO1 and p53 or FoxO1+3 [72], improves (FoxO1 and human FoxO3) chondrocytes downregulation survival also suppresses via reducing SIRT-1; proapoptotic this protein tyrosineregulates phosphatase oxidative stress 1B and (PTP1B) autophagy [73 by], modulating and also manages the signaling autophagy of FoxO and directly p53 [72], via improves deacetylation of key componentshuman chondrocytes (such as survival ATG5, via 7, reducing and 8) in proapoptotic the autophagy protein inductiontyrosine phosphatase network 1B [74 (PTP1B)]. Besides, the [73], and also manages autophagy directly via deacetylation of key components (such as ATG5, 7, decreasedand SIRT-1, 8) in the in autophagy response induction to siFoxO, network brought [74]. Besi aboutdes, the reduced decreased FoxO SIRT-1, transcriptional in response to siFoxO, activity [70]. Collectively,brought in response about reduced to ROS FoxO in chondrocytes, transcriptional FoxOsactivity mediate[70]. Collectively, antioxidant in response and autophagy to ROS in proteins through alteringchondrocytes, the SIRT-1 FoxOs mediate level to antioxidant some extent and autophagy (Figure5). proteins through altering the SIRT-1 level to some extent (Figure 5). Commented [M1]: There is a highlight in original Word file, please check if the highlight needs to be removed.

Figure 5. FoxOs increase antioxidants and further regulate multiple functions of chondrocytes. FoxOs Figure 5. FoxOs increase antioxidants and further regulate multiple functions of chondrocytes. FoxOs possibly promotepossibly proliferationpromote proliferation and block and hypertrophic block hypertrophic differentiation differentiation along withalong the with PTEN the/Akt /FoxO signaling.PTEN/Akt/FoxO FoxOs also repress signaling. apoptosis FoxOs also and repress autophagy apoptosis with and altering autophagy the with SIRT-1 altering protein the level.SIRT-1 PTEN = phosphataseprotein and level. tensin PTEN homolog = phosphatase deleted and ontensin homolog deleted ten. on chromosome ten.

Combined deletion of FoxO1, 3, and 4 in mice growth plate chondrocytes (chondrocyte triple knock-out; CTKO) significantly reduces expression levels of oxidative stress defense genes, such as Gpx3, Sepp1, and Sesn3, in the primary chondrocytes, which may elevate the levels of ROS and

Int. J. Mol. Sci. 2020, 21, x; doi: FOR PEER REVIEW www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 692 15 of 22

Table 5. Bone metabolism regulatory functions of FoxO transcription factors in chondrocytes.

FoxOs Effects on Bone Functions in Chondrocyte Mechanisms Cell/Mice Models References FoxO1 and FoxO3 up-regulated antioxidant proteins and human articular chondrocyte FoxO1, chondrocyte viability (+); autophagy-related proteins, but transfected into siFoxO1 and [70] FoxO3 chondrocyte apoptosis (-) decreased expression of siFoxO3 ADAMTS-4 and chemerin. hypertrophic zone of the growth plate (-); overall FoxO1, 3, expression of genes involved in FoxO1,3a,4f/f; Collagen2-Cre body and tail length at eight weeks of age (-); [67] and 4 redox homeostasis (+) mice; hyperkyphosis (-) total body and tail length at 1 month of age (-); height of the proliferative zone of proximal tibial growth Col2Cre-FoxO1, 3, and 4 triple FoxO1, 3, plate at P7 and 1 month (-); articular cartilage thicker chondrocyte proliferation autophagy and antioxidant defense knockout mice (Col2Cre-TKO); and 4 at 1 or 2 months of age (-); OA-like changes developed (-); cell density (+); genes (+); Prg4 expression (+) Col2Cre-FoxO1 knockout mice [68] in cartilage, synovium, and subchondral bone between 4 and 6 months of age (-) Col2Cre-FoxO3 or 4 single FoxO3 or 4 no cartilage abnormalities until 18 months of age knockout mice spontaneous cartilage degradation and OA severity in FoxO1, 3, deletion of FoxO1/3/4 in mature a surgical model or treadmill running of skeletally cell density (+); Prg4 expression (+) and 4 mice using Aggrecan-CreERT2 mature mice (-) expression level of markers specific cell apoptosis (+); for mature (aggrecan, collagen II) multipotent mesenchymal FoxO3 chondrogenic [69] and hypertrophic (collagen X) stromal cells (MSCs) differentiation (-) chondrocytes (-) Int. J. Mol. Sci. 2020, 21, 692 16 of 22

Combined deletion of FoxO1, 3, and 4 in mice growth plate chondrocytes (chondrocyte triple knock-out; CTKO) significantly reduces expression levels of oxidative stress defense genes, such as Gpx3, Sepp1, and Sesn3, in the primary chondrocytes, which may elevate the levels of ROS and promote more proliferated chondrocytes turning to pre-hypertrophic and hypertrophic chondrocytes [67,75]. Interestingly, chondrocyte-specific inactivation of the tumor suppressor gene phosphatase and tensin homolog deleted on chromosome ten (PTEN) also generated a disorganized neonatal growth plate, hyperkyphosis, and an increased total body length, which is similar to the phenotype of FoxOs CTKO mice [76]. Besides, PTEN can act as the upstream of FoxOs by regulating Akt activation [24]. Moreover, the Akt/FoxO pathway enhances chondrocyte proliferation but decreases chondrocyte maturation and cartilage matrix production [77]. Therefore, as antioxidant factors in chondrocytes, FoxOs may promote proliferation and block hypertrophic differentiation through the signaling along the PTEN/Akt/FoxO axis (Figure5). In addition, FoxO1 downregulation in chondrocytes also dramatically upregulates the expression of the inflammatory factor, chemerin, which increases chondrocyte apoptosis to some degree [70]. Through transcription factor FoxO1, pro-inflammatory cytokines TNF-α activates chondrocyte apoptosis and accelerates the loss of cartilage in diabetic fracture healing [78,79]. Moreover, TNF-α promote the chemokine expression of chondrocytes in diabetic fracture healing [80]. Hence, FoxO1 may protect diabetic fracture healing from an inflammation effect through TNF-α/chemokine. In short, by decreasing ROS accumulation during chondrogenesis, FoxOs promote proliferation and block hypertrophic differentiation along with the PTEN/Akt/FoxO signaling, as well as repress apoptosis and autophagy by altering the Sirt1 protein level. All of these demonstrate that FoxOs contribute to relieving osteoarthritis and improve the growth, development, and health of cartilage.

4. Conclusion and Perspective In summary, through FoxO deletion or overexpression in early osteoblast progenitors, osteoblasts, hematopoietic stem cells, osteoclast precursors, or chondrocytes, these sufficient and seminal papers have revealed the complicated and prominent roles of FoxO transcription factors not only in regulating bone cells function, but also in controlling the bone modeling or remodeling and preserving skeletal homeostasis. In particular, FoxOs regulate bone cell survival, cell cycle, proliferation, differentiation, autophagy, even apoptosis, and also participate in network control among different kinds of bone cells (Figure6). They are intimately involved in many bone physiological and pathological activities, such as bone development, bone remodeling, energy metabolism, aging, osteoarthritis, low bone mass in type 1 diabetes, postmenopausal osteoporosis, age-related bone loss, and myeloid . For example, FoxOs improve expression of antioxidant enzymes and maintain relatively low ROS levels to protect MSCs/HSCs from osteogenesis/osteoclastogenesis. During differentiation, ROS levels are increased due to enhanced cell metabolism. FoxOs promote osteogenesis and inhibit adipogenesis or osteoclastogenesis by decreasing ROS levels. Moreover, age-related mechanisms intrinsic to bone and oxidative stress are more proven as the protagonist of osteoporosis (including postmenopausal osteoporosis) [81]. Therefore, in bone, all the three FoxO members (FoxO1, 3, and 4) jointly mediate the redox balance of these bone cells, thereby regulating bone metabolism and also involved in osteoporosis pathology. Above all, these roles of FoxOs may help in the development of pharmacological agents, targeting FoxOs to further elucidate the treatment of bone metabolism diseases. Int. J. Mol. Sci. 2020, 21, 692 17 of 22 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 2 of 24

Figure 6. FigureThe role 6. ofThe FoxOs role inof osteoprogenitorFoxOs in osteoprogenitor cells, chondrocytes, cells, chondrocytes, osteoblasts, osteoblasts, osteocytes, osteocytes, hematopoietic stem cells,hematopoietic and osteoclast stem precursors, cells, and osteoclast as well precursors, as in the as complex well as in intercellular the complex signalingintercellular among signaling these bone cells. FoxOsamong not these only bone regulate cells. survival,FoxOs not self-renewal, only regulate cellsurvival, cycle, self-renewal, proliferation, cell dicycle,fferentiation, proliferation, autophagy, differentiation, autophagy, and apoptosis of bone cells, but also participate in the interaction among and apoptosis of bone cells, but also participate in the interaction among these bone cells. FoxO’s these bone cells. FoxO’s function in osteocytes is unknown. function in osteocytes is unknown. Before that, several questions should be paid attention to, including but not limited to the Beforefollowing: that, several Firstly, questionshow FoxO factors should balance be paid theattention positive and to, negative including regulation but not of limitedbone formation? to the following: Firstly, howOn one FoxO hand, factors FoxO1 balancefacilitates thebone positive formation andby in negativeteracting with regulation Runx2 in osteoprogenitor of bone formation? cells or On one hand, FoxO1with ATF4 facilitates in osteoblasts, bone formation while diminishing by interacting bone formation with Runx2 by mediating in osteoprogenitor the function of cells M- or with CSF/RANKL in osteoclasts. Here, FoxO1 should be an important factor for bone formation, but not ATF4 in osteoblasts,involved in redox while regulation. diminishing On the other bone hand, formation FoxOs mediate by mediating the redox the balance function in or among of M-CSF bone /RANKL in osteoclasts.cells, thereby Here, regulating FoxO1 shouldbone metabolism be an important and homeos factortasis. Therefore, for bone the formation, balance role of but FoxOs not may involved in redox regulation.depend on Onthe thebone other metabolism hand, stage FoxOs and mediatebone redox the homeostasis. redox balance Secondly, in or what among are the bone complete cells, thereby regulatingand bone precise metabolism mechanisms andof FoxOs homeostasis. performing functions Therefore, among the the balance different role kinds of of FoxOs bone cells? may For depend on the boneexample, metabolism FoxOs stage regulate and redox bone balance redox involved homeostasis. in the activities Secondly, of all whatkinds of are bone the cells. complete Then, how and precise the FoxOs mediate ROS signaling in or among bone cells? Thirdly, how FoxOs regulate bone mechanismsremodeling of FoxOs and performingpreserve skeletal functions homeostasis among during the aging different in bone? kinds For ofexample, bone cells?with aging, For example, FoxOs regulateexcessive redox osteocyte balance apoptosis involved correlates in the with activities oxidative of stress all kindsand favors of bone osteoclastogenesis, cells. Then, howwhich the FoxOs mediate ROSleads to signaling increased inbone or remodeling among bone imbalance cells? and Thirdly, bone loss. how This FoxOs results in regulate the occurrence bone of remodeling several and preservebone skeletal diseases, homeostasis especially osteoporosis. during aging Then, in does bone? FoxOs For inhibit example, osteocyte with apoptosis aging, by regulating excessive an osteocyte anti-oxidation system in osteocytes? And what is the mechanism involved therein? Is it effective to apoptosis correlates with oxidative stress and favors osteoclastogenesis, which leads to increased bone treat and prevent bone loss? In light of this evidence, more research is necessary to further illuminate remodelingthe imbalanceprecise role of and FoxOs, bone especially loss. This for th resultse development in the occurrence of new therapeutic of several approaches. bone diseases, especially osteoporosis.Author Then, Contributions: does FoxOs X.M. and inhibit Y.T. conceived osteocyte the concept apoptosis of this manuscript by regulating and wrote an the anti-oxidation first draft version system in osteocytes?of it, And which what was then is theequally mechanism developed by involved P.S., C.Y., X.L therein?., X.W., Y.G., Is itS.P., eff A.R.W.,ective A.Q. to treat (Abdul and Qadir) prevent and A.Q. bone loss? In light of(Airong this evidence,Qian) revised moreand proofread research the manuscript; is necessary all authors to further have read illuminate and approved the the precise final submitted role of FoxOs, manuscript. especially for the development of new therapeutic approaches. Funding: This work was funded by the National Natural Science Foundation of China (nos. No.81801871) and Author Contributions:China PostdoctoralX.M. Science and Foundation Y.T. conceived (nos. No. the 2017M613210). concept of this manuscript and wrote the first draft version of it, which wasConflicts then of equally Interest: developedThe authors declare by P.S., no C.Y.,conflict X.L., of interest. X.W., Y.G., S.P., A.R.W., A.Q. (Abdul Qadir) and A.Q. (Airong Qian) revised and proofread the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded by the National Natural Science Foundation of China (nos. No.81801871) and China Postdoctoral Science Foundation (nos. No. 2017M613210). Conflicts of Interest: The authors declare no conflict of interest. Int. J. Mol. Sci. 2020, 21, 692 18 of 22

Abbreviations

FoxOs Forkhead box class O family member proteins LPS Lipopolysaccharide TNF-α Tumor necrosis factor α MSCs Mesenchymal stem cells Runx2 Runt-related transcription factor 2 ALP Alkaline phosphatase OCN Osteocalcin CFU Colony forming unit PPARγ Peroxisome proliferator-activated receptor γ PPREs PPAR-response elements PCNA Proliferating cell nuclear antigen ROS Reactive oxygen species FABP4 Fatty acid-binding protein 4 4-HNE 4-hydroxynonenal ATF4 Activating transcription factor 4 BFR Bone formation rate HSC Hematopoietic stem cell Gpx-1 Glutathione peroxidase 1 M-CSF Macrophage-colony stimulating factor RANK Receptor activator of NF-κ B RANKL Receptor activator of NF-κ B ligand BMMCs Bone marrow mononuclear cells NFATc1 Nuclear factor of activated T cells 1 Akt SIRT-1 Sirtuin 1 TRAP Tartrate-resistant acid phosphatase MAPKs Mitogen-activated protein kinases NF-κB Nuclear factor kappa-B HO-1 Hemeoxygenase-1 Mst 1 Mammalian Sterile 20-like kinase 1 OPG Osteoprotegerin AP-1 Activator protein 1 PI3K phosphoinositide 3-kinase OA Osteoarthritis PTP1B Protein tyrosine phosphatase 1B CTKO Chondrocyte triple knock-out PTEN Phosphatase and tensin homolog deleted on chromosome ten

References

1. Carter, M.E.; Brunet, A. FOXO transcription factors. Curr. Biol. 2007, 17, R113–R114. [CrossRef][PubMed] 2. Avila-Flores, A.; Arranz-Nicolas, J.; Merida, I. Transcriptional Activity of FOXO Transcription Factors Measured by Luciferase Assays. In FOXO Transcription Factors; Springer: Berlin, Germany, 2019; pp. 91–102. 3. Genin, E.C.; Caron, N.; Vandenbosch, R.; Nguyen, L.; Malgrange, B. Concise review: Forkhead pathway in the control of adult neurogenesis. Stem Cells 2014, 32, 1398–1407. [CrossRef][PubMed] 4. Jensen, K.S.; Binderup, T.; Jensen, K.T.; Therkelsen, I.; Borup, R.; Nilsson, E.; Multhaupt, H.; Bouchard, C.; Quistorff, B.; Kjær, A. FoxO3A promotes metabolic adaptation to hypoxia by antagonizing Myc function. EMBO J. 2011, 30, 4554–4570. [CrossRef][PubMed] 5. Tuteja, G.; Kaestner, K.H. SnapShot: Forkhead transcription factors I. Cell 2007, 130, 1160. [CrossRef] 6. Calnan, D.; Brunet, A. The foxo code. Oncogene 2008, 27, 2276. [CrossRef] 7. Henderson, S.T.; Johnson, T.E. daf-16 integrates developmental and environmental inputs to mediate aging in the . Curr. Biol. 2001, 11, 1975–1980. [CrossRef] Int. J. Mol. Sci. 2020, 21, 692 19 of 22

8. Webb, A.E.; Brunet, A. FOXO transcription factors: Key regulators of cellular quality control. Trends Biochem. Sci. 2014, 39, 159–169. [CrossRef] 9. Eijkelenboom, A.; Burgering, B.M. FOXOs: Signalling integrators for homeostasis maintenance. Nat. Rev. Mol. Cell Biol. 2013, 14, 83. [CrossRef] 10. Kim, H.-N.; Iyer, S.; Ring, R.; Almeida, M. The role of FoxOs in bone health and disease. In Current Topics in Developmental Biology; Elsevier: Amsterdam, The Netherlands, 2018; Volume 127, pp. 149–163. 11. Bullock, M. FOXO factors and breast cancer: Outfoxing endocrine resistance. Endocr. Relat. Cancer 2016, 23, R113–R130. [CrossRef] 12. Wang, Y.; Zhou, Y.; Graves, D.T. FOXO transcription factors: Their clinical significance and regulation. BioMed Res. Int. 2014, 2014, 925350. [CrossRef] 13. De Brachène, A.C.; Demoulin, J.-B. FOXO transcription factors in cancer development and therapy. Cell. Mol. Life Sci. 2016, 73, 1159–1172. [CrossRef][PubMed] 14. Tsitsipatis, D.; Klotz, L.-O.; Steinbrenner, H. Multifaceted functions of the forkhead box transcription factors FoxO1 and FoxO3 in skin. Biochim. Biophys. Acta. Gen. Subj. 2017, 1861, 1057–1064. [CrossRef][PubMed] 15. Sun, Z.; da Fontoura, C.S.; Moreno, M.; Holton, N.E.; Sweat, M.; Sweat, Y.; Lee, M.K.; Arbon, J.; Bidlack, F.B.; Thedens, D.R. FoxO6 regulates Hippo signaling and growth of the craniofacial complex. PLoS Genet. 2018, 14, e1007675. [CrossRef][PubMed] 16. Chen, D.; Gong, Y.; Xu, L.; Zhou, M.; Li, J.; Song, J. Bidirectional regulation of osteogenic differentiation by the FOXO subfamily of Forkhead transcription factors in mammalian MSCs. Cell Prolif. 2019, 52, e12540. [CrossRef] 17. Fu, Z.; Tindall, D. FOXOs, cancer and regulation of apoptosis. Oncogene 2008, 27, 2312. [CrossRef] 18. Iyer, S.; Ambrogini, E.; Bartell, S.M.; Han, L.; Roberson, P.K.; de Cabo, R.; Jilka, R.L.; Weinstein, R.S.; O’Brien, C.A.; Manolagas, S.C. FOXOs attenuate bone formation by suppressing Wnt signaling. J. Clin. Investig. 2013, 123, 3409–3419. [CrossRef] 19. Brent, M.M.; Anand, R.; Marmorstein, R. Structural basis for DNA recognition by FoxO1 and its regulation by posttranslational modification. Structure 2008, 16, 1407–1416. [CrossRef] 20. Murtaza, G.; Khan, A.K.; Rashid, R.; Muneer, S.; Hasan, S.M.F.; Chen, J. FOXO transcriptional factors and long-term living. Oxid. Med. Cell. Longev. 2017, 2017, 3494289. [CrossRef] 21. Almeida, M. Unraveling the role of FoxOs in bone—insights from mouse models. Bone 2011, 49, 319–327. [CrossRef] 22. Teixeira, C.C.; Liu, Y.; Thant, L.M.; Pang, J.; Palmer, G.; Alikhani, M. Foxo1, a novel regulator of osteoblast differentiation and skeletogenesis. J. Biol. Chem. 2010, 285, 31055–31065. [CrossRef] 23. Komori, T. Regulation of bone development and protein genes by RUNX2. Cell Tissue Res. 2010, 339, 189. [CrossRef][PubMed] 24. Van der Horst, A.; Burgering, B.M. Stressing the role of FoxO proteins in lifespan and disease. Nat. Rev. Mol. Cell Biol. 2007, 8, 440. [CrossRef][PubMed] 25. Siqueira, M.F.; Flowers, S.; Bhattacharya, R.; Faibish, D.; Behl, Y.; Kotton, D.N.; Gerstenfeld, L.; Moran, E.; Graves, D.T. FOXO1 modulates osteoblast differentiation. Bone 2011, 48, 1043–1051. [CrossRef][PubMed] 26. Ambrogini, E.; Almeida, M.; Martin-Millan, M.; Paik, J.-H.; DePinho, R.A.; Han, L.; Goellner, J.; Weinstein, R.S.; Jilka, R.L.; O’Brien, C.A. FoxO-mediated defense against oxidative stress in osteoblasts is indispensable for skeletal homeostasis in mice. Cell Metab. 2010, 11, 136–146. [CrossRef] 27. Klotz, L.-O.; Steinbrenner, H. Cellular adaptation to xenobiotics: Interplay between xenosensors, reactive oxygen species and FOXO transcription factors. Redox Biol. 2017, 13, 646–654. [CrossRef][PubMed] 28. Armoni, M.; Harel, C.; Karni, S.; Chen, H.; Bar-Yoseph, F.; Ver, M.R.; Quon, M.J.; Karnieli, E. FOXO1 represses peroxisome proliferator-activated receptor-gamma1 and -gamma2 gene promoters in primary adipocytes. A novel paradigm to increase insulin sensitivity. J. Biol. Chem. 2006, 281, 19881–19891. [CrossRef] 29. Fan, W.; Imamura, T.; Sonoda, N.; Sears, D.D.; Patsouris, D.; Kim, J.J.; Olefsky, J.M. FOXO1 transrepresses peroxisome proliferator-activated receptor γ transactivation, coordinating an insulin-induced feed-forward response in adipocytes. J. Biol. Chem. 2009, 284, 12188–12197. [CrossRef] 30. Muller, F.L.; Lustgarten, M.S.; Jang, Y.; Richardson, A.; Van Remmen, H. Trends in oxidative aging theories. Free Radic. Biol. Med. 2007, 43, 477–503. [CrossRef] Int. J. Mol. Sci. 2020, 21, 692 20 of 22

31. Almeida, M.; Han, L.; Martin-Millan, M.; O’Brien, C.A.; Manolagas, S.C. Oxidative stress antagonizes Wnt signaling in osteoblast precursors by diverting β-catenin from T cell factor-to forkhead box O-mediated transcription. J. Biol. Chem. 2007, 282, 27298–27305. [CrossRef] 32. Almeida, M.; Han, L.; Martin-Millan, M.; Plotkin, L.I.; Stewart, S.A.; Roberson, P.K.; Kousteni, S.; O’Brien, C.A.; Bellido, T.; Parfitt, A.M. Skeletal involution by age-associated oxidative stress and its acceleration by loss of sex steroids. J. Biol. Chem. 2007, 282, 27285–27297. [CrossRef] 33. Bennett, C.N.; Longo, K.A.; Wright, W.S.; Suva, L.J.; Lane, T.F.; Hankenson, K.D.; MacDougald, O.A. Regulation of osteoblastogenesis and bone mass by Wnt10b. Proc. Natl. Acad. Sci. 2005, 102, 3324–3329. [CrossRef][PubMed] 34. Babij, P.; Zhao, W.; Small, C.; Kharode, Y.; Yaworsky, P.J.; Bouxsein, M.L.; Reddy, P.S.; Bodine, P.V.; Robinson, J.A.; Bhat, B. High bone mass in mice expressing a mutant LRP5 gene. J. Bone Miner. Res. 2003, 18, 960–974. [CrossRef][PubMed] 35. Schneider, C.; Porter, N.A.; Brash, A.R. Routes to 4-hydroxynonenal: Fundamental issues in the mechanisms of lipid peroxidation. J. Biol. Chem. 2008, 283, 15539–15543. [CrossRef][PubMed] 36. Almeida, M.; Ambrogini, E.; Han, L.; Manolagas, S.C.; Jilka, R.L. Increased lipid oxidation causes oxidative stress, increased peroxisome proliferator-activated receptor-γ expression, and diminished pro-osteogenic Wnt signaling in the skeleton. J. Biol. Chem. 2009, 284, 27438–27448. [CrossRef] 37. Kang, S.; Bennett, C.N.; Gerin, I.; Rapp, L.A.; Hankenson, K.D.; MacDougald, O.A. Wnt signaling stimulates osteoblastogenesis of mesenchymal precursors by suppressing CCAAT/enhancer-binding protein α and peroxisome proliferator-activated receptor γ. J. Biol. Chem. 2007, 282, 14515–14524. [CrossRef] 38. Okamura, M.; Kudo, H.; Wakabayashi, K.-i.; Tanaka, T.; Nonaka, A.; Uchida, A.; Tsutsumi, S.; Sakakibara, I.; Naito, M.; Osborne, T.F. COUP-TFII acts downstream of Wnt/β-catenin signal to silence PPARγ and repress adipogenesis. Proc. Natl. Acad. Sci. USA 2009, 106, 5819–5824. [CrossRef] 39. Sharma, C.; Pradeep, A.; Wong, L.; Rana, A.; Rana, B. Peroxisome proliferator-activated receptor γ activation can regulate β-catenin levels via a proteasome-mediated and adenomatous polyposis coli-independent pathway. J. Biol. Chem. 2004, 279, 35583–35594. [CrossRef] 40. Atashi, F.; Modarressi, A.; Pepper, M.S. The role of reactive oxygen species in mesenchymal stem cell adipogenic and osteogenic differentiation: A review. Stem Cells Dev. 2015, 24, 1150–1163. [CrossRef] 41. Rached, M.-T.; Kode, A.; Xu, L.; Yoshikawa, Y.; Paik, J.-H.; DePinho, R.A.; Kousteni, S. FoxO1 is a positive regulator of bone formation by favoring protein synthesis and resistance to oxidative stress in osteoblasts. Cell Metab. 2010, 11, 147–160. [CrossRef] 42. Clemens, T.L.; Karsenty, G. The osteoblast: An insulin target cell controlling glucose homeostasis. J. Bone Miner. Res. 2011, 26, 677–680. [CrossRef] 43. Yang, S.; Xu, H.; Yu, S.; Cao, H.; Fan, J.; Ge, C.; Fransceschi, R.T.; Dong, H.H.; Xiao, G. Foxo1 mediates insulin-like growth factor 1 (IGF1)/insulin regulation of osteocalcin expression by antagonizing Runx2 in osteoblasts. J. Biol. Chem. 2011, 286, 19149–19158. [CrossRef][PubMed] 44. Rached, M.-T.; Kode, A.; Silva, B.C.; Jung, D.Y.; Gray, S.; Ong, H.; Paik, J.-H.; DePinho, R.A.; Kim, J.K.; Karsenty, G. FoxO1 expression in osteoblasts regulates glucose homeostasis through regulation of osteocalcin in mice. J. Clin. Investig. 2010, 120, 357–368. [CrossRef] 45. Behl, Y.; Siqueira, M.; Ortiz, J.; Li, J.; Desta, T.; Faibish, D.; Graves, D.T. Activation of the acquired immune response reduces coupled bone formation in response to a periodontal pathogen. J. Immunol. 2008, 181, 8711–8718. [CrossRef] 46. Bigarella, C.L.; Li, J.; Rimmelé, P.; Liang, R.; Sobol, R.W.; Ghaffari, S. FOXO3 transcription factor is essential for protecting hematopoietic stem and progenitor cells from oxidative DNA damage. J. Biol. Chem. 2017, 292, 3005–3015. [CrossRef][PubMed] 47. Yalcin, S.; Zhang, X.; Luciano, J.P.; Mungamuri, S.K.; Marinkovic, D.; Vercherat, C.; Sarkar, A.; Grisotto, M.; Taneja, R.; Ghaffari, S. Foxo3 is essential for the regulation of ataxia telangiectasia mutated and oxidative stress-mediated homeostasis of hematopoietic stem cells. J. Biol. Chem. 2008, 283, 25692–25705. [CrossRef] [PubMed] 48. Tothova, Z.; Kollipara, R.; Huntly, B.J.; Lee, B.H.; Castrillon, D.H.; Cullen, D.E.; McDowell, E.P.; Lazo-Kallanian, S.; Williams, I.R.; Sears, C. FoxOs are critical mediators of hematopoietic stem cell resistance to physiologic oxidative stress. Cell 2007, 128, 325–339. [CrossRef] Int. J. Mol. Sci. 2020, 21, 692 21 of 22

49. Buck, D.W.; Dumanian, G.A. Bone biology and physiology: Part I. The fundamentals. Plast. Reconstr. Surg. 2012, 129, 1314–1320. [CrossRef] 50. Boyce, B. Advances in the regulation of osteoclasts and osteoclast functions. J. Dent. Res. 2013, 92, 860–867. [CrossRef] 51. Wang, Y.; Dong, G.; Jeon, H.H.; Elazizi, M.; La, L.B.; Hameedaldeen, A.; Xiao, E.; Tian, C.; Alsadun, S.; Choi, Y. FOXO1 mediates RANKL-induced osteoclast formation and activity. J. Immunol. 2015, 194, 2878–2887. [CrossRef] 52. Nakashima, T.; Hayashi, M.; Takayanagi, H. New insights into osteoclastogenic signaling mechanisms. Trends Endocrinol. Metab. 2012, 23, 582–590. [CrossRef] 53. Sugatani, T.; Hruska, K.A. Akt1/Akt2 and mammalian target of rapamycin/Bim play critical roles in osteoclast differentiation and survival, respectively, whereas Akt is dispensable for cell survival in isolated osteoclast precursors. J. Biol. Chem. 2005, 280, 3583–3589. [CrossRef][PubMed] 54. Kawamura, N.; Kugimiya, F.; Oshima, Y.; Ohba, S.; Ikeda, T.; Saito, T.; Shinoda, Y.; Kawasaki, Y.; Ogata, N.; Hoshi, K. Akt1 in osteoblasts and osteoclasts controls bone remodeling. PLoS ONE 2007, 2, e1058. [CrossRef] 55. Bartell, S.M.; Kim, H.-N.; Ambrogini, E.; Han, L.; Iyer, S.; Ucer, S.S.; Rabinovitch, P.; Jilka, R.L.; Weinstein, R.S.; Zhao, H. FoxO proteins restrain osteoclastogenesis and bone resorption by attenuating H 2 O 2 accumulation. Nat. Commun. 2014, 5, 3773. [CrossRef][PubMed] 56. Lee, N.K.; Choi, Y.G.; Baik, J.Y.; Han, S.Y.; Jeong, D.-w.; Bae, Y.S.; Kim, N.; Lee, S.Y. A crucial role for reactive oxygen species in RANKL-induced osteoclast differentiation. Blood 2005, 106, 852–859. [CrossRef][PubMed] 57. Tan, P.; Guan, H.; Xie, L.; Mi, B.; Fang, Z.; Li, J.; Li, F. FOXO1 inhibits osteoclastogenesis partially by antagnozing MYC. Sci. Rep. 2015, 5, 16835. [CrossRef] 58. Ferber, E.C.; Peck, B.; Delpuech, O.; Bell, G.P.; East, P.; Schulze, A. FOXO3a regulates reactive oxygen metabolism by inhibiting mitochondrial gene expression. Cell Death Differ. 2012, 19, 968. [CrossRef] 59. Peck, B.; Ferber, E.C.; Schulze, A. Antagonism between FOXO and MYC regulates cellular powerhouse. Front. Oncol. 2013, 3, 96. [CrossRef] 60. Ke, K.; Safder, M.; Sul, O.-J.; Kim, W.-K.; Suh, J.-H.; Joe, Y.; Chung, H.-T.; Choi, H.-S. Hemeoxygenase-1 maintains bone mass via attenuating a redox imbalance in osteoclast. Mol. Cell. Endocrinol. 2015, 409, 11–20. [CrossRef] 61. Kim, H.-N.; Han, L.; Iyer, S.; de Cabo, R.; Zhao, H.; O’Brien, C.A.; Manolagas, S.C.; Almeida, M. Sirtuin1 suppresses osteoclastogenesis by deacetylating FoxOs. Mol. Endocrinol. 2015, 29, 1498–1509. [CrossRef] 62. Reszka, A.A.; Halasy-Nagy, J.M.; Masarachia, P.J.; Rodan, G.A. Bisphosphonates act directly on the osteoclast to induce caspase cleavage of mst1 kinase during apoptosis A link between inhibition of the mevalonate pathway and regulation of an apoptosis-promoting kinase. J. Biol. Chem. 1999, 274, 34967–34973. [CrossRef] 63. Jang, S.-W.; Yang, S.-J.; Srinivasan, S.; Ye, K. Akt phosphorylates MstI and prevents its proteolytic activation, blocking FOXO3 phosphorylation and nuclear translocation. J. Biol. Chem. 2007, 282, 30836–30844. [CrossRef] [PubMed] 64. Boyle, W.J.; Simonet, W.S.; Lacey, D.L. Osteoclast differentiation and activation. Nature 2003, 423, 337. [CrossRef] 65. Domazetovic, V.; Marcucci, G.; Iantomasi, T.; Brandi, M.L.; Vincenzini, M.T. Oxidative stress in bone remodeling: Role of antioxidants. Clin. Cases Miner. Bone Metab. 2017, 14, 209. [CrossRef][PubMed] 66. Akasaki, Y.; Hasegawa, A.; Saito, M.; Asahara, H.; Iwamoto, Y.; Lotz, M. Dysregulated FOXO transcription factors in articular cartilage in aging and osteoarthritis. Osteoarthr. Cartil. 2014, 22, 162–170. [CrossRef] [PubMed] 67. Eelen, G.; Verlinden, L.; Maes, C.; Beullens, I.; Gysemans, C.; Paik, J.-H.; DePinho, R.A.; Bouillon, R.; Carmeliet, G.; Verstuyf, A. Forkhead box O transcription factors in chondrocytes regulate endochondral bone formation. J. Steroid Biochem. Mol. Biol. 2016, 164, 337–343. [CrossRef] 68. Matsuzaki, T.; Alvarez-Garcia, O.; Mokuda, S.; Nagira, K.; Olmer, M.; Gamini, R.; Miyata, K.; Akasaki, Y.; Su, A.I.; Asahara, H. FoxO transcription factors modulate autophagy and proteoglycan 4 in cartilage homeostasis and osteoarthritis. Sci. Transl. Med. 2018, 10, eaan0746. [CrossRef] 69. Djouad, F.; Bony, C.; Canovas, F.; Fromigue, O.; Reme, T.; Jorgensen, C.; Noel, D. Transcriptomic analysis identifies Foxo3A as a novel transcription factor regulating mesenchymal stem cell chrondrogenic differentiation. Cloning Stem Cells 2009, 11, 407–416. [CrossRef] Int. J. Mol. Sci. 2020, 21, 692 22 of 22

70. Akasaki, Y.; Alvarez-Garcia, O.; Saito, M.; Caramés, B.; Iwamoto, Y.; Lotz, M.K. FoxO transcription factors support oxidative stress resistance in human chondrocytes. Arthritis Rheumatol. 2014, 66, 3349–3358. [CrossRef] 71. Fujita, N.; Matsushita, T.; Ishida, K.; Kubo, S.; Matsumoto, T.; Takayama, K.; Kurosaka, M.; Kuroda, R. Potential involvement of SIRT1 in the pathogenesis of osteoarthritis through the modulation of chondrocyte gene expressions. J. Orthop. Res. 2011, 29, 511–515. [CrossRef] 72. Salminen, A.; Kaarniranta, K. SIRT1: Regulation of via autophagy. Cell. Signal. 2009, 21, 1356–1360. [CrossRef] 73. Gagarina, V.; Gabay, O.; Dvir-Ginzberg, M.; Lee, E.J.; Brady, J.K.; Quon, M.J.; Hall, D.J. SirT1 enhances survival of human osteoarthritic chondrocytes by repressing protein tyrosine phosphatase 1B and activating the insulin-like growth factor receptor pathway. Arthritis Rheum. 2010, 62, 1383–1392. [CrossRef][PubMed] 74. Ng, F.; Tang, B.L. Sirtuins’ modulation of autophagy. J. Cell. Physiol. 2013, 228, 2262–2270. [CrossRef] [PubMed] 75. Morita, K.; Miyamoto, T.; Fujita, N.; Kubota, Y.; Ito, K.; Takubo, K.; Miyamoto, K.; Ninomiya, K.; Suzuki, T.; Iwasaki, R. Reactive oxygen species induce chondrocyte hypertrophy in endochondral ossification. J. Exp. Med. 2007, 204, 1613–1623. [CrossRef][PubMed] 76. Ford-Hutchinson, A.F.; Ali, Z.; Lines, S.E.; Hallgrímsson, B.; Boyd, S.K.; Jirik, F.R. Inactivation of Pten in osteo-chondroprogenitor cells leads to epiphyseal growth plate abnormalities and skeletal overgrowth. J. Bone Miner. Res. 2007, 22, 1245–1259. [CrossRef][PubMed] 77. Rokutanda, S.; Fujita, T.; Kanatani, N.; Yoshida, C.A.; Komori, H.; Liu, W.; Mizuno, A.; Komori, T. Akt regulates skeletal development through GSK3, mTOR, and FoxOs. Dev. Biol. 2009, 328, 78–93. [CrossRef] [PubMed] 78. Kayal, R.A.; Siqueira, M.; Alblowi, J.; McLean, J.; Krothapalli, N.; Faibish, D.; Einhorn, T.A.; Gerstenfeld, L.C.; Graves, D.T. TNF-α mediates diabetes-enhanced chondrocyte apoptosis during fracture healing and stimulates chondrocyte apoptosis Through FOXO1. J. Bone Miner. Res. 2010, 25, 1604–1615. [CrossRef] [PubMed] 79. Alblowi, J.; Kayal, R.A.; Siqueria, M.; McKenzie, E.; Krothapalli, N.; McLean, J.; Conn, J.; Nikolajczyk, B.; Einhorn, T.A.; Gerstenfeld, L. High levels of tumor necrosis factor-α contribute to accelerated loss of cartilage in diabetic fracture healing. Am. J. Pathol. 2009, 175, 1574–1585. [CrossRef] 80. Alblowi, J.; Tian, C.; Siqueira, M.F.; Kayal, R.A.; McKenzie, E.; Behl, Y.; Gerstenfeld, L.; Einhorn, T.A.; Graves, D.T. Chemokine expression is upregulated in chondrocytes in diabetic fracture healing. Bone 2013, 53, 294–300. [CrossRef] 81. Manolagas, S.C. From estrogen-centric to aging and oxidative stress: A revised perspective of the pathogenesis of osteoporosis. Endocr. Rev. 2010, 31, 266–300. [CrossRef]

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).